Monitoring and controlling pH is critical to many industrial processes, perhaps none more so than biopharmaceutical manufacturing, which depends so closely on sensitive animal or plant cells. pH control is essential to optimizing biopharma’s key quality parameters, and ensuring final product quality and yield.

However, this control can prove elusive, both up and downstream. Metabolic changes within micro-organisms can change the pH of their environment, as can changes in process conditions. In addition, sensor drift can result when sensors are fouled, or improperly calibrated after equipment cleaning and sterilization, affecting readings. This article will review the reasons why pH control is so important to biopharmaceutical operations, highlight some best practices for achieving that control, and briefly discuss how pH control can enable Quality by Design (QbD) of biopharmaceuticals.

In downstream biopharma applications, during chromatography steps, pH (along with ionic strength) is a key property within the buffered mobile phase that has a critical impact on protein purification. Interactions between the mobile and stationary phases can affect chromatographic performance, and thereby the separation profile between product and impurities. For example, product recovery, purity and throughput can be adversely affected by variability in buffer preparation and critical parameters such as pH during production-scale bioprocessing.

In addition, pH must be carefully monitored during the formulation process prior to fill/finish operations.

Typical upstream applications involve the development, optimization and implementation of either a fermentation process—utilizing living organisms such as a yeast, bacteria, or fungi to produce a pharmaceutically active product—or cell culture, a process in which a mammalian cell is grown to produce the active product.

Microbial fermentation processes are normally shorter in duration (2-7 days) than cell culture processes (typically about two weeks for fed batch processes). Extracellular pH is an important bioprocessing parameter in the culture of mammalian cells for the production of diagnostic and therapeutic proteins, viral vaccines, and cells for somatic and gene therapies. It must be optimized along with other culture parameters, such as media formulation and oxygen tension, to obtain the desired cellular and product characteristics. Extracellular pH must also be effectively controlled in order to ensure the quality and consistency of the culture outcome, be it cells or a cell-derived product.

pH regulation can be particularly challenging because organisms alter the pH of their environment through metabolic activity. Typical effects of cell metabolism on pH include:

1.    Production of organic acids (lactic, acetic, butyric, etc.) as fermentative end products of carbohydrate fermentation.
2.    Production of ammonia from nitrogen-containing compounds such as amino acids, particularly when the nitrogen to carbon ratio in the medium components is higher than the ratio typical of biomass (1:4), which tends to raise pH.
3.    Production of CO2 and consequent elevation of carbonic acid in the medium.
4.    Consumption of the anion of an organic acid such as succinate would elevate pH.

Therefore, pH management and control is an important aspect of designing cell culture media on both the laboratory and industrial scale. Many research programs require that an investigator grow cells in laboratory culture, and the development of appropriate culture media requires consideration of pH control while minimizing osmolarity changes and ensuring consistent pH profiles from run to run.

Culture Control

Most cell culture media contain components intended to provide pH buffering, which is desirable because the growth of many cells is restricted to narrow limits of pH. Control of pH in bioprocessing typically involves the use of an acid source (typically CO2 gas) and a base source (typically sodium carbonate) to maintain pH within ± 0.03pH unit from a set point.

Measurement of pH in a Chinese hamster ovary (CHO) cell culture, monitored over time, shows that pH changes are negligible during the early phases of cell growth when cell density is low. This is followed by a rapid decline in pH over a period of approximately 60 hours when no further changes are observed [1].

Culture pH affects several cellular functions and properties including cell proliferation, cell differentiation, cell metabolism, intracellular pH, how ammonia affects cells, protein glycosylation, protein synthesis, antibody production, enzyme mRNA levels , enzyme secretion, glutamine synthesis and glutamine decomposition. Since variations in culture pH affect several cellular functions and properties, effective pH control is necessary in industrial-scale mammalian cell culture.

One recent study demonstrated that, in a culture of typical mammalian cells in protein-free medium (both mouse myeloma, NSO, and CHO), both the number of viable cells and the production rate of the culture were higher when cultured at pH 7.0 versus 7.3. It was concluded that a simple three-tenths of a pH unit difference appeared to provide for a doubling of the productivity of the culture. Further, it was demonstrated that a reduction of culture pH generally results in reduced specific glucose utilization and lactate accumulation rates for the model cell line when maintained within the permissible range for cell growth.

A lower specific glucose utilization rate was deemed beneficial, since it reduced the quantity of glucose to be added as part of the fed-batch process, simplifying feed formulation and addition, while a lower specific lactate accumulation rate reduced the overall lactate concentration and thereby limited the amount of alkali required to control culture pH, simplifying process operation and improving process robustness [2].